U.S. patent number 5,032,407 [Application Number 07/004,077] was granted by the patent office on 1991-07-16 for gene transfer using transformed, neodetermined, embryonic cells.
This patent grant is currently assigned to Ohio University Edison Animal Biotechnology Center. Invention is credited to Barbara J. Corn, Michael A. Reed, Thomas E. Wagner.
United States Patent |
5,032,407 |
Wagner , et al. |
July 16, 1991 |
Gene transfer using transformed, neodetermined, embryonic cells
Abstract
This invention is directed to a method for the preparation of
carrier cells capable of delivering exogenous genetic material to a
particular tissue of the body by means of embryonic cells competent
to develop into that tissue, and essentially only that tissue, said
cells bearing the exogenous genetic material. The preferred carrier
cells are mesodermal cells of the yolk sac or embryonic forebrain
or midbrain cells, and the desired genetic material is preferably
introduced into the cells by in vitro transformation with an
amphotrophic retroviral vector.
Inventors: |
Wagner; Thomas E. (Athens,
OH), Reed; Michael A. (Athens, OH), Corn; Barbara J.
(Athens, OH) |
Assignee: |
Ohio University Edison Animal
Biotechnology Center (Athens, OH)
|
Family
ID: |
21709025 |
Appl.
No.: |
07/004,077 |
Filed: |
January 16, 1987 |
Current U.S.
Class: |
800/23; 514/44R;
424/582; 424/520 |
Current CPC
Class: |
C12N
5/0605 (20130101); C12N 5/0603 (20130101); A61K
48/00 (20130101); C12N 2510/00 (20130101) |
Current International
Class: |
A61K
48/00 (20060101); C12N 5/06 (20060101); A61K
035/00 (); C12N 015/00 (); C12N 005/00 () |
Field of
Search: |
;435/172.3,240.2
;800/1,2,DIG.2 ;424/520,582,93 ;935/62 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
4497796 |
February 1985 |
Salser et al. |
|
Other References
Anderson, French W., "Prospects for Human Gene Therapy", Science
226:401 (1984). .
"Differential Susceptibility of Mouse Trophoblast and Embryonic
Tissue to Immune Cell Lysis", Jenkinson and Billington,
Transplantation, 18:286-88 (1974). .
"Transplantation of Fetal Hematopoietic Stem Cells in Utero: The
Creation of Hamatopoietic Chimeras", Flake, et al., Science,
233:776-78 (1986). .
"Introduction of a Selectable Gene into Primitive Stem Cells
Capable of Long-Term Reconstitution of the Hemopoietic System of
W/W.sup.v Mice", Dick, et al., Cell, 42:71-79 (1985). .
"Retrovirus Transfer of a Bacterial Gene into Mouse Haematopoietic
Progenitor Cells", Joyner, et al., Nature, 305:556-559 (1983).
.
Studies on the Immunobiology of Mouse Fetal Membranes: "The Effect
of Cell-Mediated Immunity of Yolk Sac Cells in Vitro", J. Reprod.
Fert., 41:403-412 (1974). .
Billington and Jenkinson, "Antigen Expression During Early Mouse
Development", Balls and Wild, The Early Development of Mammals,
219-232, (1975). .
Ritter, "Early Differentiation of the Lymphoid System", Balls and
Wild, The Early Development of Mammals, 359-372 (1975). .
"Factors Regulating Yolk Sac Hematopoiesis in Diffusion Chambers:
Various Types of Sera, Cyclophosphamide, Irradiation and Long-Term
Culture", Weinberg and Stohlman, Jr., Exp. Hematol., 5:374-384
(1977). .
"An in vitro Morphological Study of the Mouse Visceral Yolk Sac and
Possible Yolk Sac Immunocyte Precursors", Cell Tissue Res., 113-119
(1977). .
"Hemopoiesis and Blood Vessels in Human Yolk Sac", Hesseldahl and
Larsen, Acta. Anat. 78:271-291 (1971). .
"Initial Growth of Transplanted E11 Fetal Cortex and Spinal Cord in
Adult Rat Spinal Cord", Bernstein, et al., Brain Research
343:336-345 (1985). .
"Establishment in Culture of Pluripotential Cells from Mouse
Embryos", Evans and Kaufman, et al., Nature, 292:154-156 (1981).
.
"Some Aspects of Tissue Interaction in Vitro", Auerbachm
Epithelical-Mesenchymal Interactions, Chap. 13, pp. 200-7 (1968).
.
"Evidence for the Time of Appearance of H-2 Antigens in Mouse
Development," Patthey and Edidin, Transplantation, 15:211-214
(1973). .
"Demonstration of Permanent Factor-Dependent Multipotential
(Erythroid/Neutrophil/Basophil) Hematopoietic Progenitor Cell
Lines", Greenberger, et al., P.N.A.S. (U.S.A.), 80:2931-35 (1983).
.
"Introduction of New Genetic Material into Pluripotent
Haematopoietic Stem Cells of the Mouse", Williams, et al., Nature,
310:476-80 (1984). .
"Gene Expression in Mice After High Efficiency Retroviral-Mediated
Gene Transfer", Eglitis, et al., Science 230;1395-98 (1985). .
"Amphotropic Retrovirus Vector Transfer of the v-ras Oncogene to
Human Hematopoietic and Stromal Cells in Continuous Bone Marrow
Cultures", Rothstein, et al., Blood, 65:744-752 (1985). .
"Fetal Liver, A Source for Hemopoietic Reconstitution without
GHVD?", Heit, et al., Biology of Bone Marrow Transplantation,
507-517 (Academic Press; 1980). .
"Fetal Bone Grafts do not Elicit Allograft Rejection Because of
Protecting Anti-Ia Alloantibodies", Segal, et al., Transplantation,
28:88-95 (1979). .
"Transgenesis by Means of Blastocyst-Derived Embryonic Stem Cell
Lines", Gossler et al., PNAS (U.S.A.) 83:9065-69 (Dec. 1986). .
"Totipotent Hematopoietic Stem Cells: Normal Self-Renewal and
Differentiation after Transplantation between Mouse Fetuses",
Fleischman, et al., Cell, 30:351-59 (1982). .
"Murine Yolk Sac Hematopoiesis Studied with the Diffusion Chamber
Technique" Symann, et al., Exp. Hematol., 6:749-59 (1978). .
Lovell-Badge et al., Cold Spring Harbor Symp. Quant. Biol., vol. L,
pp. 707-712 (1985), Cold Spring Harbor Lab., New York. .
Gossler et al., Proc. Natl. Acad. Sci. 83:9065-9 (1986). .
Kuehn et al., Nature 326:295 (1987)..
|
Primary Examiner: Weimar; Elizabeth C.
Assistant Examiner: Chambers; Jasemine C.
Attorney, Agent or Firm: Cooper; Iver P.
Claims
We claim:
1. A method of providing tissue-specific expression of exogenous
genetic material is selected tissues of a recipient mammal which
comprises:
(a) providing carrier cells capable of selectively delivering said
exogenous genetic material to said tissues, said cells selected
from the group consisting of embryonic yolk sac cells, embryonic
midbrain cells and embryonic forebrain cells, all of which are
derived from a donor mammal of the same species, said cells also
having been transformed in vitro with said exogenous genetic
material and
(b) introducing said cells into said recipient mammal in a manner
permitting them to differentiate into said tissues.
2. The method of claim 1, where the carrier cells are mesodermal
cells of the yolk sac which are competent to develop into
hematopoietic stem cells, and the cells are introduced
intravenously.
3. The method of claim 1, where the carrier cells are embryonic
forebrain or midbrain cells.
4. The method of claim 1, where the cells are transformed with a
retroviral vector carrying the genetic material.
5. The method of claim 4, where the retroviral vector is
amphotrophic.
6. The method of claim 1 where the animal is immunocompetent and
the introduction of the carrier cells does not result in an immune
response.
7. The method of claim 1, said cells being essentially
histocompatible with a host of the same species as the mammal from
which the embryonic cells were derived.
8. The method of claim 1 in which the embryonic cells provided have
been immortalized.
9. The method of claim 1, wherein the cells of the tissue are
deficient in said genetic material.
10. The method of claim 1, where the cells are transformed by
microinjecting them with the genetic material.
11. The method of claim 1 wherein the carrier cells are at a
sufficiently early stage of development so that introduction of the
carrier cells into an immunocompetent mammal does not result in an
immune response to said carrier cells.
12. The method of claim 11 wherein the mammal is
immunocompetent.
13. The method of claim 1 wherein the carrier cells are yolk sac
cells derived from yolk sacs extracted from an embryonic donor
mammal prior to the formation of blood islands visible at 12X
magnification.
14. A process for producing a chimeric mammal characterized by the
presence of heterologous genetic material in selected tissues of
said mammal, which comprises introducing heterologous genetic
material into said tissues by the method of claim 1.
15. The method of claim 1 wherein the carrier cells are provided in
a non-enzymatically disaggregated form.
Description
BACKGROUND OF THE INVENTION
The invention relates to the use of early embryonic cells to
deliver genetic material into a fully formed animal.
It is often desirable to confer upon an animal a particular genetic
trait.
It is possible to remove bone marrow cells from the animal,
transform them with a vector carrying the desired gene, and
reimplant the transformed cells. Generally speaking, the
transformed cells are given a competitive advantage. For example,
the animal may be irradiated to partially or completely destroy the
normal marrow, thus providing the transformed marrow cells with a
vacant ecological niche. See, e.g., Joyner, et al., Nature
(London), 305: 556 (1983). Clearly, this damage to the host is
undesirable in a practical genetic delivery system.
Salser, U.S. Pat. Nos. 4,396,601 and 4,497,796 removed bone marrow
cells from mice, cotransformed them with DNA including HSV DNA and
the marker DHFR gene, and selected for cells resistant to
methotrexate. These drug-resistant cells were injected into
irradiated mice treated with methotrexate. Preferably, the bone
marrow cell population used was one rich in hematopoietic stem
cells. Salser does not teach use of cells removed from a mammalian
embryo, and Salser severely stressed the recipient mice to give the
modified cells a selective advantage.
Wagner et al, WO 82/04443 placed exogenous material into the
pronucleus of a zygote. They teach that the zygote should be
transformed as soon as possible after fertilization. We transform
embryonic cells at a considerably later stage of development.
Mintz and Illmensee, PNAS 72:3585 (1975) injected teratocarcinoma
(embryonal carcinoma, EC) cells into mouse blastocysts, obtaining
mosaic mice. The teratocarcinoma cells proved to be developmentally
totipotent, developing into a variety of normal tissues. This
method cannot be used to deliver genes into specific tissues
because the developmental course of these cells is uncertain.
EC-like totipotent cells (EK cells) have also been obtained from
culturing ICM cells of normal mouse embryos removed on day 2.5.
Evans and Kaufman, Nature, 292:154 (1981).
We have found a convenient method for delivering genes into
specific tissues of an animal which does not require extraction of
any cells from the animal. Rather, we transform post-gastrular
embryonic cells, which, besides being easier to culture, may be
selected to be both (1) predestined to develop into the target
tissue, and (2) essentially non-immunogenic.
One method known for the transformation of explanted cells involves
use of a retroviral vector. See Vande Woude, U.S. Pat. No.
4,405,712.
When a retrovirus infects a cell, its RNA genome acts as a template
for the reverse transcription of the viral genetic information into
a double strand of DNA. This DNA molecule, now called a provirus,
integrates into the genome of the host. Retroviral RNA is
synthesized from the proviral sequence by the host's own RNA
polymerase, and some of this RNA is translated into viral proteins.
Under the instruction of the packaging sequence (called psi in the
Moloney murine leukemia virus studies), the RNA-protein core of the
virus is packaged into a glycoprotein envelope, and the resulting
viral particle buds off from the cell into the medium (where it may
find and infect other cells).
Mann, et al., Cell, 33:153 (1983) developed a cell line, known as
psi-2, which is a line of NIH 3T3 cells with a permanently
integrated helper virus. The helper virus, psi-minus, corresponds
to the MoMLV with the psi sequence deleted by BalI-PstI cleavage.
The psi-2 cells produce viral particles only when transformed by a
retroviral vector bearing the psi sequence.
Cone and Mulligan, PNAS 81:6349 (1984), of the same research group,
later developed an improved packaging cell line, psi-AM. This .cell
line was developed by transforming NIH 3T3 cells with a psi-minus
chimera of an amphotrophic retrovirus (4070A). This amphotrophic
murine retrovirus could infect non-murine hosts, including human
and monkey cells.
Both psi-2 and psi-AM cells are readily available in the scientific
community.
Joyner et al., supra, used an MoMLV retroviral vector to transfer a
neomycin resistance gene into mouse hematopoietic progenitor cells.
Williams, et al., Nature (London) 310: 476 (1984) used MSV DHFR-NEO
transformed psi-2 cells to transfer neomycin resistance to
co-cultivated bone marrow cells. See also Greenberger, et al.,
PNAS, 80:2931 (1983); Dick, et al., Cell, 42:71 (August 1985);
Rubinstein, et al., 81:7137 (1984); Rothstein, et al., Blood,
65:744 (1985).
The above references teach retroviral transformation of "primitive
but committed" non-embryonic cells. "Primitive" is a relative term,
and these hematopoietic bone marrow stem cells are much further
advanced in development than are the embryonic cells of the
immediate post-gastrular stage ("neodetermined"), and therefore are
likely to be less pluripotent and less histocompatible.
Verma, et al. , in Tumor Viruses and Cell Differentiation, 251
(Scolnick and Levine, eds., 1983) and Miller, et al., PNAS (USA)
80: 4709 (1983) and Science, 225: 630 (1984) also describe use of
retroviral vectors in gene therapy.
Genes may also be inserted by other techniques, such as calcium
phosphate-mediated DNA uptake. Wigler, et al., Cell, 11: 223
(1977). To assure survival and proliferation of the transformed
cells, powerful selection systems, such as DHFR/methotrexate, are
used to inhibit untransformed cells. Carr, et al., Blood, 62: 180
(1983); Cline, et al., Nature, 284: 422 (1980). Without such
selection, the efficiency of this procedure is presently too low to
affect the recipient's condition significantly.
While Hammer, et al., Nature (London) 311: 65 (1984) has used
microinjection of an RGH gene to correct dwarfism in the mouse, the
technique is too labor intensive to be commercially practicable,
even if other difficulties were overcome.
Lipid vesicles containing exogenous DNA have been injected into the
tail vein of mice, so transformation occurs in vivo. Szoka, U.S.
Pat. No. 4,394,448.
Kiester, Jr., Science 86, at 33 (March 1986) reports on research in
which rat fetal brain tissue was grafted intraocularly into adult
rats.
Jacob, EP Appl. 178,220 used a retroviral vector to confer G418
resistance on three embryonal carcinoma cell lines. He teaches
implanting genetically engineered embryos into the uterus of a
female mammal where it may naturally develop into a transgenic
infant. This is to be distinguished from the present invention, in
which engineered embryonic cells are injected into the bloodstream,
or the corresponding tissue of the recipient. Jacob also teaches
removing bone-marrow cells from a postnatal animal, transforming
the cells, and returning them to the same animal.
Heit et al., in The Biology of Bone Marrow Transplantation, 507-517
(1980) suggested that fetal liver cells could be used for
hematopoietic reconstitution without a graft versus host reaction.
Mouse fetal liver cells have been microinjected into the placental
circulation and thereby introduced into a recipient fetus. While
this technique permits donor hematopoietic cells to become
competitively established without ablation or irradiation of the
recipient, it is dependent on the immunological immaturity of both
donor cells and recipient. Fleischman, et al., Cell, 30:351-359
(1982); Flake, et al., Science, 233:776-778 (1986). Segal, et al.,
Transplantation, 28:88-95 (1979) suggests a mechanism whereby fetal
bone grafts may escape host rejection in immunocompetent hosts.
Japanese application 61-81743 is said to relate to "a mature,
non-human animal containing germ and somatic cells transformed by
an activated tumor sequence, which was introduced into the animal
or its ancestor during the fetal stage."
Yolk sac cells which produce an embryonic variant hemoglobin have
been injected into irradiated adult mice of another strain and the
surviving mice were found to be producing, in part, the donor adult
type of hemoglobin. Auerbach, in EPITHELIAL-MESENCHYMAL
INTERACTIONS, ch. 13 (1968).
SUMMARY OF THE INVENTION
This invention relates to the preparation of carrier cells capable
of delivering genetic material to a particular tissue of the body
by means of embryonic cells competent to develop into that tissue,
and essentially only that tissue, said cells bearing the desired
genetic material. The preferred carrier cells are mesodermal cells
of the yolk sac or embryonal forebrain or midbrain cells, and the
desired genetic material is preferably introduced into the cells by
transformation with an amphotrophic retroviral vector. The yolk sac
carrier cells are introduced intravenously. Preferably, the carrier
cells may be introduced into an immunocompetent host without
provoking an immune response.
Mammalian development may be divided into three distinct stages:
the zygote, from fertilization to cleavage; the embryo, from
cleavage to the formation of all somites; and the fetus, from the
formation of the last somite until birth. This invention takes
advantage of the unique properties of embryonic cells after their
course of development is determined, but before they have lost
immunoincompetency or the ability to proliferate rapidly.
An embryo begins with fertilization of an egg by a sperm. The
fertilized egg is called a zygote. The unicellular zygote develops
by successive mitotic divisions into a multicellular complex, the
morula. The cells of the morula move outward to form a blastula.
The daughter cells are called blastomeres, and are typically
arrayed as a spherical layer, the blastoderm, surrounding a cavity,
the blastocoele.
Gastrulation is the process by which the blastoderm differentiates
into an ectoderm, a mesoderm, and an endoderm. The ectoderm will
develop into the skin and nervous system; the mesoderm, into the
muscular, skeletal, circulatory and excretory systems; and the
endoderm, into the digestive system. For a number of organisms,
"fate maps" have been ccnstructed which show the normal
developmental fate of each part of the blastula.
In the early gastrula stage, the prospective potency of the neural
area of the ectoderm is such that if cells are transplanted to
another area, they can develop into not only epidermis, but also
mesodermal or endodermal tissues. At the end of gastrulation, a
transplanted piece of presumptive neural tissue will differentiate
as brain or spinal cord in whatever part of the embryo it is
placed. Clearly, the surrounding tissues affected the development
of the transplant. These inducing tissues act by releasing chemical
inducers. By cultivating inductor tissues in suitable media, it is
possible to produce "conditioned media" which contain effective
amounts of the inducing substances.
The narrowing of the potency of the embryonic tissue is called
determination. The range of developmental possibilities still open
to a piece of tissue is its competency.
The first stage of development of the mammalian zygote is repeated
cleavage into a solid mass of cells, the morula. The morula
develops into a second structure, the blastocyst, having a distinct
outer layer (the trophoblast) and an inner cell mass. The
trophoblast enlarges and detaches from one side of the inner cell
mass to create a yolk sac cavity, and the surface of the inner cell
mass differentiates to form a hypoblast.
The trophoblast acts to attach the developing embryo to the walls
of the uterus, a process known as implantation. The trophoblast
cells become the placenta of the developing organism. The inner
cell mass is destined to differentiate into ectodermal, mesodermal
and endodermal tissues. Nonetheless, all of the ICM cells of the
blastocyst are essentially totipotent, that is, they can develop
into any tissue of the body. Evans and Kaufman, Nature, 292:154
(1981). It is only after gastrulation that their destiny is
determined to some degree.
For one view of cell lineages in the mouse embryo, see FIG. 3 in
Gardner and Papaiioannou, in THE EARLY DEVELOPMENT OF MAMMALS 107
(Balls and Wild, eds, Cambridge U:1975).
It is known that when completely undifferentiated cells of the
blastula or morula are transplanted into a developed animal, they
produce tumors. Id.
These totipotent, tumorigenic cells are of no value as genetic
delivery systems. However, we have discovered that it is
advantageous to transplant cells which have reached that stage of
specialization at which they have become committed to a particular
sequence of development, or lineage. Such cells may be used to
deliver genetic material, or its expression products, into a
particular tissue of the body, including blood cells.
The cells are transformed with genetic material of interest,
transplanted into a host, and allowed to develop into the target
tissue.
While it is necessary to use cells which have matured to the point
of losing totipotency, overly mature cells will be rejected by the
host. Consequently, it is desirable to use cells which have just
lost totipotency. Such cells also retain the ability to colonize,
thus facilitating their delivery to the target tissue.
Certain cells of the yolk sac offer particular advantages as
tissue-specific genetic delivery means.
Unlike the cells of the embryo, the cells of the yolk sac develop
into only a small number of different tissues. Among those tissues
is the erythropoietic system, which includes the red and white
blood cells, and the tissue of the veins, arteries and capillaries.
Thus, by day 8 in the development of the mouse, mesodermal cells in
the yolk sac have formed blood islets. The cells of the blood
islets differentiate, the peripheral cells becoming the endothelium
of the future blood vessels, and the central cells becoming first
mesenchymal cells and then the red and white blood cells. The blood
islands establish communications to form a circulatory network,
which is extended into the embryo proper.
For the mouse, a standard reference work is Rugh, The Mouse: Its
Reproduction and Development. According to this reference,
gastrulation occurs at day 5.5. The mesoderm appears at day 6.5 as
mesenchymal cells, and essentially separates the other two primary
germ layers by day 7. The yolk sac is also formed by this time.
Organogenesis begins at day 7.5 and the neural groove is formed by
day 8. The circulatory system derives from blood islands,
aggregations of mesenchymal cells in the mesoderm of the
splanchnopleure. Blood cells are formed as early as day 7. By day
8, early forming blood islands and lacunae leading to vessels may
be seen in the yolk sac. By day 8.5, blood islands are plentiful,
and blood vessels are beginning to appear. By day 9, capillaries
are also plentiful.
In the ectoderm, the prosencephalon (forebrain) and mesencephalon
(midbrain) are both apparent by day 8. The neural crest develops on
day 9.
Preferably, the yolk sacs are extracted prior to formation of
visible (12X) blood islands.
Billington and Jenkinson, working with cells of the yolk sac of
10-14 day mouse embryos, found that these cells ex pressed both H-2
and non-H2 (major and minor histocompatibility) antigens. The work
of Meyner (1973) and Patthey & Edidin (1973) cited by
Billington and Jenkinson, reported that H2 antigens first appear on
day 7 embryos, but the latter suggested that these antigens did not
make an appearance in utero until day 9 or later. See THE EARLY
DEVELOPMENT OF MAMMALS 219 (Balls and Wild, eds., Cambridge
U.:1975).
We believe that the persistence of G-418-resistant yolk sac cells
for three weeks after injection indirectly shows their ability to
escape immune rejection. By contrast, research with bone marrow
cells has depended on the use of immunocompromised hosts.
In another embodiment of this invention, embryonic yolk sac cells
which do not necessarily contain exogenous DNA are introduced into
an immunodeficient or hematopoieticdeficient host for purposes of
hematopoietic reconstitution.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an autoradiograph showing the transfer of neomycin
resistance to the bone marrow cell population using transformed,
neodetermined cells.
FIG. 2 is a map of the pLJ vector.
DETAILED DESCRIPTION OF THE INVENTION
EXAMPLE 1
Cells of the yolk sac were surgically removed from a mouse embryo
at day 7 or 8, physically disaggregated by drawing through a 21
gauge needle, and plated on culture media (Hams F12, 10% FCS, 10
ug/ml gentamicin). They aggregated to form spheres of yolk sac
cells which attached themselves to the culture vessels. Physical
disaggregation is preferred over enzymatic disaggregation using
trypsin and collagenase.
Five different types of cells were identified in isolated yolk
sacs: (1) a small, rounded cell; (2) a squamous epithelial cell;
(3) a star-shaped cell appearing intermediate between (3a)
fibroblast and (3b) epithelial cells; (4) a short, compact,
dome-shaped fibroblast cell; and (5) a large, flat, basophilic
epithelial cell believed to be the precursor of blood and lymphatic
vessel cells. The last four types are believed to be mesodermal
cells.
Mesodermal cells may be separated from endodermal cells by
treatment with glycine according to Dziadek, Exper. Cell Res.
133:383 (1981).
EXAMPLE 2
The four mesodermal cell types were cloned by single cell dilution
into conditioned media. The medium is preferably one used to
support a mixed culture of yolk sac cells for 24 hours (see Example
1).
EXAMPLE 3
It is particularly advantageous to immortalize these primary cell
lines. Immortalized cell lines may be produced from any of the five
yolk sac cell types.
We have prepared immortalized mixed cultures, as well as an
immortalized pure culture of yolk sac cell type (3). The primary
cell line is grown to confluency in the original medium, thus
selecting for cells which can grow under hypoxic conditions.
Curatolo, et al., In Vitro, 20:597 (1984). Trypsin is used to
dislodge the cells, and they are diluted 1:2 into fresh media.
After 5-10 such passages, a crisis is reached at which most of the
cells are inactive while a few, often polyploidal, are dividing
rapidly. The latter are the immortalized cells, which may then be
cloned and cultured.
These immortalized cell lines have the advantage that they may be
maintained indefinitely in vitro.
EXAMPLE 4
In another embodiment of this invention, cells are surgically
removed from the embryonic fore- and midbrain of a day 8 mouse
embryo, disaggregated and cultured according to Example 1, and
cloned according to Example 2. These cells have been immortalized
by the method of Example 3.
EXAMPLE 5
The cells may now be transformed with the desired genetic material.
While direct transformation is acceptable, it is preferable to use
a vector, particularly a retroviral vector. The efficiency of
retroviral transfer is higher.
Psi-2 cells are transformed with a modified pLJ retroviral vector
(N2). In the pLJ vector, the viral genes (gag, pol, env) are
deleted, but the packaging sequence and the LTR sequences (which
are necessary for integration) remain. The modified pLJ vector used
bears a transcriptional unit comprising an SV40 promoter, a
neomycin resistance gene, and an SV40 terminator. See Eglitis,
Science, 230:1395 (December 1985); Joyner, et al., nature, 305:556
(1983).
The psi-2 cells are placed in a medium containing modified pLJ
retroviral particles. After several days cultivation, transformed
cells are selected by killing untransformed cells with the
mammalian cell toxin G418 [Gibco]. The expression product of the
neomycin resistance gene phosphorylates and thereby inactivates
G418, thus protecting the transformed cells. The cells are grown in
the G418 media for about three weeks. A concentration of 500 ug/ml
G418 is preferred.
In one embodiment of the invention, the yolk sac cells are
cultivated in the supernatant from the transformed psi-2 cell
culture. This supernatant contains the recombinant retroviral
particles. The disadvantage of this approach is that the particles
do not remain viable indefinitely.
Preferably, the yolk sac cells (pre-treated with polybrene) are
co-cultivated with the transformed psi-2 cells, so they are
continually exposed to the recombinant retroviral particles. If
this is done, then there must be a way of distinguishing the
transformed yolk sac cells from the transformed psi-2 cells, since
both will carry neomycin resistance.
Mitomycin C destroys the ability of psi-2 cells to replicate,
without hindering their production of retroviral particles. Psi-2
cells were treated with mitomycin C (concentration 2 ug/10.sup.6
cells) at 50% confluency. While they cannot divide, they will still
produce virus for several weeks. The yolk sac cells are added to
the treated psi-2 cell culture. The viral particles produced by the
psi-2 cells transfect the yolk sac cells. Eventually, after several
subdivisions of the culture, the psi-2 cells die off, leaving only
the yolk sac cells. Transformed yolk sac cells are then
distinguished on the basis of resistance to G418.
Alternatively, psi-AM cells may be transfected with an amphotrophic
retroviral vector and used to supply viral particles to the
neodetermined carrier cells. Psi-AM cells are preferable to psi-2
cells in that they can carry a virus which is not
species-specific.
EXAMPLE 6
Five day old newborn mice were intracranially injected with
immortalized embryonic fore- and midbrain cells prepared according
to Example 4. These cells had been transformed, as taught in
Example 5, by a Neo.sup.R -carrying retroviral vector in the
presence of psi-2 helper cells. The mice were sacrificed after
development to adulthood (five weeks old) and their brain cells
were cultured.
EXAMPLE 7
We have demonstrated that by the method of this invention,
heterologous genetic material (a neomycin resistance gene) was
transferred to a specific tissue and measurably expressed by the
recipient animal without ablation of the target tissue.
The tail vein of a 4 week mouse (ICR strain) was injected with
250-500,000 transformed (YS) cells. The animal was sacrificed and
its spleen and bone marrow cells are examined for the presence of
neomycin resistance genes.
The spleen cells (about 10.sup.6 /well) were placed in suspension
culture (RPMI 1640, 10% FCS, 25 mM HEPES buffer, 10 ug/ml
gentamicin) with a T cell mitogen (phytohemagglutin, 25 ug/ml,
Burroughs-Wellcome) in the presence of three different
concentrations (500 ug/ml; 250 ug/ml; 125 ug/ml) of G418 for three
days. The cells were assayed for the ability to replicate by
measuring their uptake of tritiated thymidine. Tritiated thymidine
(6.7 Ci/mmole) was added on the second day and the cells were
harvested on the third day. Response of mice injected with YS cells
(transformed with neo.sup.R bearing retrovirus) was compared with
that of mice injected with psi-2 cells, and of mice which did not
receive any cellular injection. Spleen cells from transformed YS
cell injected animals cultured in 125 ug/ml G418 exhibited an
activity which was 50.9% of that shown by spleen cells of control
animals not exposed to G418. G418-treated spleen cells from psi-2
cell injected animals and control animals had activities of 40.3%
and 39.7%, respectively. Thus, the injection of neo.sup.R
-transformed YS cells into the tail vein resulted in the expression
of increased G418 resistance in the spleen.
In the table below, the counts are given, which are reflective of
the cells' ability to replicate.
______________________________________ Control Mouse mean
______________________________________ pHA, m 51.2 6070.4 40.0
129.7 cells, m 629.0 213.4 1252.0 1323.3 Cells, PHA 2524.3 3193 7
2284.8 2158.9 2540 500 536.6 313.1 372.1 457.0 419.7 16.5% 250
757.3 619.9 1957.8 738.2 1018.1 40% 125 1406.6 863.8 1119.0 645.7
1008.7 39.7% ______________________________________ Psi-2 inj.
mouse mean ______________________________________ pHA, m 76.2 82.7
104.3 73.0 cells, m 866.6 1154.5 693.3 863.4 Cells, PHA 10656.1
8156.3 8743.3 7092.4 8666.7 500 1113.9 691.1 1269.5 759.5 958.5 11%
250 2761.5 1817.7 3331.9 1949.6 2465 28.4% 125 3665.6 3012.1 4430.2
2859.4 3491.8 40.3% ______________________________________ YS inj.
mouse mean ______________________________________ pHA, m 577.6 96.0
38.6 cells, m 951.7 889.7 648.6 Cells, PHA 5161.5 6951.7 4502.0
5530.4 500 1201.1 954.6 1201.0 1118.9 20% 250 2119.8 1164.1 2621.6
1968.5 35.5% 125 4277.5 1688.1 2488.1 2817.9 50.9%
______________________________________
We also observed that bone marrow cells from transgenic mice were
able to resist G418, but we did not quantify the resistance.
EXAMPLE 8
Three weeks after injection of transformed YS cells, the bone
marrow cells from the treated animal were lysed, and the lysate was
analyzed by the method of Eglitis, et al., Science, 230: 1395, 1397
(Dec. 20, 1985) and Reiss, et al., Gene 30:211 (1984). Lysates were
electrophoresed on a nondenaturing polyacrylamide gel. The gel was
overlayed with agarose containing kanamycin at 25 ug/ml and 2 nM
gamma-.sup.32 P-ATP (greater than 5000 Ci/mmol). The gel was
blotted with Whatman P81 phosphocellulose paper. After washing to
remove the ATP, autoradiography (FIG. 1) revealed the presence of
the radiolabeled gamma phosphate group if the neomycin resistance
enzyme (a phosphotransferase) transferred it to the kanamycin
substrate.
Referring to FIG. 1, lanes 1 and 10 are psi-2. Lane 2 is cultured
mouse yolk sac cells (untransfected); lane 3, bone marrow of
transformed YS cell-injected animals; lane 5, the bone marrow
control; lane 6, the spleen control; lane 7, brain cells
transfected with psi-2 supernate; lane 8, brain cells pre-treated
with polybrene and transfected with psi-2 supernate; and lane 9,
transformed YS cells pre-treated with polybrene and transfected
with psi-2 supernate. (Polybrene is used to enhance retroviral
infection). It will be seen from FIG. 1 that lanes 1, 3, 9 and 10
are heavily marked. The light bands in other lanes are indicative
of basal levels of phosphotransferase activity.
This evidences the transfer of a genetic trait to the bone marrow
cell population by a cellular genetic delivery system.
EXAMPLE 9
Two days after injection, the YS cells were found in the bone
marrow. On the third day they were found in the spleen. By one week
after injection, the YS cells could no longer be distinguished
morphologically from the surrounding tissue. However, as noted in
Examples 6 and 7, bone marrow and spleen cells still exhibited
neomycin resistance, even several weeks after injection. This
suggests that the injected cells engaged in "catch-up"
differentiation, rapidly differentiating into the target
tissue.
EXAMPLE 10
The uterus of a pig was removed by hysterectomy at day 20, yielding
17 embryos. The yolk sacs revealed visible islets. Yolk sac cells
were isolated and cultured by the previously described techniques,
both with and without glycine treatments. The same five YS cell
types were observable. These have been maintained in mixed
cultures.
Pig forebrain and midbrain cells were also cultured.
It is believed that it may be preferable to use day 18 embryos,
since the day 20 pig embryos appeared to be more advanced in
development than the day 8 mouse embryos.
Considerable literature exists on comparative embryology which may
be used to identify appropriate embryos to use in developing
genetic delivery systems for other animals. Thus, for the yolk sac
cell approach, one would look for an embryo with a fully developed
yolk sac in which blood islets, though extant, are not visible at
low magnification.
Thus, according to Tiedemann, Cell Tiss. Res., 173:109 (1976) the
yolk sac of the cat is formed by the inner endodermal lining, by a
vascular mesenchyme, and by a mesotheliuim on the outer face. In
the cat, the mesenchyme is the site of blood islands or
intravascular hematopoietic foci from the 14th until about the 38th
day. Thus, one would try to culture yolk sac cells of 12-15 day cat
embryos.
For the intracranial delivery approach, one would look for an
embryo in which the neural fold is closed (or virtually so) and the
fore-, mid, and hindbrains are evident. Thus day 8 in the mouse is
comparable to about day 19 in man.
The term "animal", as used herein, includes humans. However, it is
recognized that special legal and ethical considerations apply to
human gene therapy. Human yolk sac cells may be obtained from a
human abortus.
The term "transgenic animal" is used herein in its broad sense to
include all animals in which at least some somatic cells contain
heterologous DNA deliberately introduced into the cells. It is of
course feasible to deliver new genetic material into germ cells as
well as somatic cells. The term "chimeric animal" is used herein to
refer to animals on which the new genetic material is found in some
but not all cells. The term "tissue-specific chimeric animal"
indicates that the new genetic material is found is some tissues
and not in others.
There is no limitation on the gene which may be transferred by the
method of this invention.
Growth hormone genes may be used to enhance growth rate, increase
the efficiency of food utilization, increase lactation, or reduce
fat on carcasses. The gonadotropin releasing hormone gene may be
used for biosterilization. Synthetic genes encoding antigenic
proteins may be used to assure heightened immune response.
Lymphokine genes may have value in enhacing resistance to viruses,
tumors and other challenges. Gonadotropin genes may be used to
enhance ovulation and increase fertility. Genes regulating fatty
acid synthetase or lipase production may be used to affect the
lipid content of animal products. The genes transferred may be of
genomic, cDNA, synthetic or mixed origin, and of natural or
modified sequence.
Any embryonic tissue may be used to deliver genetic material to
tissue of the same lineage in a target animal. It is recognized
that the term "embryo", strictly speaking, does not include the
yolk sac. However, for the purpose of these specifications and
claims, the terms "embryo" or "embryonic tissue" are intended to
include all prefetal cells derived from the ICM of the blastocyst,
including the yolk sac.
It is not necessary to use cells of a single cell type as the
genetic delivery system. Thus, one could use a mixed culture of all
the yolk sac cells, pure cultures of any of the primary yolk sac
cell types, or immortalized mixed or pure cell lines.
By selecting embryonic tissue of appropriate competency, it is
possible to achieve any desired degree of specificity in targeting
the tissues of the recipient animal. For example, one might target
all ectodermal tissues, or merely the neural cells.
Reference to cells "derived" from embryonic cells is intended to
encompass in vitro cell cultures of embryonic cells and the result
of development after introduction of these cultured cells into a
recipient animal, but not cells of a postnatal animal derived from
embryonic cells by normal development in vivo.
While retroviral introduction of exogenous DNA into the embryonic
carrier cells is preferred, the DNA may also be introduced by
microinjection. It is also within the contemplation of this
invention that the exogenous DNA be microinjected into the early
embryonic cells (such as the one cell embryo) which will develop
into the preferred yolk sac carrier cells of this invention. Also,
it is possible to microinject an embryo with exogenous DNA, implant
the embryo and permit it to develop into a transgenic mammal (all
of whose cells are transformed with the exogenous DNA), breed the
mammal to obtain already transformed embryonic cells, and isolate
and culture the carrier (e.g., yolk sac, brain) cells of this
daughter embryo.
The following cell lines were deposited under the Budapest Treaty
with the American Type Culture Collection on Dec. 10, 1986:
______________________________________ CRL 9289 MBR CL1 A clonal
line of mouse brain fibroblast cells. CRL 9290 MBRP - A mixed
cultre of mouse brain fibroblast cells. CRL 9291 MYS CL1 A clonal
line of mouse yolk sac cells of type 3, subtyped "3a" because of
its fibroblast character. CRL 9292 MYS CL2 A clonal line of mouse
yolk sac cells of type 3, subtyped "3b" because of its epithelial
character. CRL 9293 MYSP - A mixed culture of yolk sac cells.
______________________________________
The deposit of these lines should not be constructed as a license
to make, use or sell the subject matter claimed herein.
The cell lines deposited hereunder, and cell lines derived
therefrom by mutation or otherwise, are of value as carrier
cells.
* * * * *